ZNFs: zinc finger families

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ZNFszinc finger families
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Zinc finger proteins

• Zinc finger proteins were first discovered as
  transcription factors.
• Zinc finger proteins are among the most abundant
  proteins in eukaryotic genomes.
• Their functions are extraordinarily diverse
• include DNA recognition,
• RNA packaging,
• transcriptional activation,
• regulation of apoptosis,
• protein folding and assembly, and
• lipid binding.
• Zinc finger structures are as diverse as their
  functions.

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Zinc finger family subfamilies with common
DBD-structure

• TFIIIA - prototype and founder member
• Zinc fingers Zn-structured domains binding DNA
• classical C2H2-fingers
• Nuclear receptors
• GATA-factors
• LIM domains
• GAL4-related factors
• Nucleocapsid proteins
• TFIIS
• RING finger
• PKC CRD

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Examples
C4-type Zif fra GATA-1
C2H2-type Zif fra Sp1 (3.fngr)
Zn
LIM-domain type Zif fra ACRP
PKC-type Zif
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Alignments
C-C-H-H
C-C-C-C
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The C2H2 subfamily
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Classical TFIIIA-related zinc fingers n x
Zn-C2H2

• History Xenopus TFIIIA the first isolated and
  cloned eukaryotic TF
• Function activation of 5S RNA transcription
  (RNAPIII)
• Rich source accumulated in immature Xenopus
  oocyttes as storage particles TFIIIA5S RNA
  ( 15 of total soluble protein)
• Purified 1980, cloned in 1984
• Mr 38 600, 344 aa
• Primary structure TFIIIA
• Composed of repeats 9x 30aa minidomains 70aa
  unique region C-trm
• Each minidomain conserved pattern of 2Cys2His
• Hypothesis each minidomain structured around a
  coordinated zinc ion (senere bekreftet)

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Zinc finger proteins

• Finger-like i 2D
• Not in 3D

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Common features of TFIIIA-related zinc fingers

• Consensus for each finger FXCX2-5CX3FX5FX2HX2-5H
• Number of fingers in related factors varies 2-37
• Number of members exceptionally high
• S.cerevisiae genome 34 C2H2 zinc fingers
• C.elegans genome 68 C2H2 zinc fingers
• Drosophila genome 234 C2H2 zinc fingers
• Humane genom 564 C2H2 zinc fingers, (135 C3HC4
  zinc finger)
• We now recognize the classical C2H2 zinc finger
  as the first member of a rapidly expanding family
  of zinc-binding modules.

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3D structure of the classical C2H2-type of zinc
fingers

• Each finger a minidomain with ???-structure
• each finger an independent module
• Several fingers linked together by fleksible
  linkers
• First 3D structure the 3-finger Zif268 (mouse)
• DNA interaction in Zif268
• major groove contact through ?-helix in ???
• recognition of base triplets
• aa in three positions responsible for sequence
  recognition -1, 3 and 6 (rel. til ?-helix)
• Simpel one-to-one pattern (contact aa - baser) ?
  can a recognition code be defined ??
• DNA interaction in GLI and TTK differs
• different phosphate contact
• distortion of DNA
• finger 1 without DNA contact

D N A
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The Zif268 prototype

• Finger 2 from Zif268
• including the two cysteine side chains and two
  histidine side chains that coordinate the zinc
  ion
• DNA-recognition residues
• indicated by the numbers identifying their
  position relative to the start of the recognition
  helix

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Three fingers in Zif268

• Zif268 - first multi-finger structure
• recognition of base triplets

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Recognition code?

• The DNA sequence of the Zif268 site is colorcoded
  to indicate base contacts made by each finger.

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Structure of the six-finger TFIIIADNA complex

• In a multi-finger protein some fingers contact
  base pairs and some will not, but rather function
  as bridges
• Fingers 123, separated by typical linkers, wrap
  smoothly around the major groove like those of
  Zif268
• In contrast, fingers 456 form an open, extended
  structure running along one side of the DNA. Of
  these, only finger 5 makes contacts with bases in
  the major groove. The flanking fingers, 4 and 6,
  appear to serve primarily as spacer elements.

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Linker connecting the fingers also important

• Linker between fingers
• Half of the known C2H2 zinc finger proteins
  contain a highly conserved linker of sequence
  TGEKP that connects adjacent fingers.
• Function of linker
• The linker is dynamically disordered in the free
  protein, but adopts well-defined structure with
  restricted backbone flexibility upon binding to
  DNA.
• DNA-induced helix capping - diffusing Zif ?
  docked Zif
• WT1 - variant linker forms ? Zifs with different
  function
• WT1 two splice variants (KTS) with an insertion
  or (KTS) without in the TGEKP linker between the
  3. and 4. zinc fingers.
• Modification of linker in vivo can have profound
  physiological consequences.
• Frasier syndrome is caused by mutation that
  prevents the KTS isoform.
• The KTS isoform binds DNA with high affinity and
  regulates transcriptionin contrast, the KTS
  variant binds DNA weakly and associates
  preferentially with the splicing machinery,where
  it may interact with RNA.
• KTS insertion increases linker flexibility,
  abrogates binding of 4. finger to DNA.

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C2H2-finger variations
Zinc finger structures are as diverse as their
functions.
Variations on the classical Cys 2 His 2 zinc
finger. (a) Classical TFIIIA-type zinc
finger (b) N-terminal zinc finger of SWI5 and
(c) BIR2 domain of XIAP.
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Zinc finger engineering

• Effort focused on the design of novel C2H2 zinc
  finger proteins
• that can specifically target unique binding sites
  within the human genome.
• applications as probes and may ultimately prove
  valuable for human gene therapy.
• Challenge to achieve the high binding affinity
  and specificity
• 1-2-3 Zif increase binding affinity 1000-fold for
  each additional finger. Only modest improvements
  in affinity occur gt3 Zif
• Design of dimerized Zif-dimers or triplets that
  recognize 10 bp with high affinity.
• Wolffes company

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Example

• Design ZFPs de novo that will bind to specific
  targeted DNA sequences.
• ZFPs were designed to regulate the endogenous
  gene encoding vascular endothelial growth
  factor-A (Vegfa). Expression of these new ZFPs in
  vivo led to induced expression of the protein
  VEGF-A, stimulation of angiogenesis and
  acceleration of experimental wound healing.
• Vegfa-activating ZFP expression induces
  angiogenesis in the mouse ear.
• These data establish that specifically designed
  transcription factors can regulate an endogenous
  gene in vivo and evoke a potentially therapeutic
  biophysiologic effect.

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Unsolved problems

• Topological problem with a factor that is wrapped
  around DNA
• 3 fingers dekker en full turn of DNA
• krysning of minor groove nødvendig når Number of
  fingers gt3
• RNA and DNA binding
• i TFIIIA finger 1-3 DNA-binding, 4-6 RNA-binding

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Kjernereceptors 2xC4
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Nuclear receptors 2xZn-C4

• Large family where DBD binds two Zn through a
  tetraedrical pattern of Cys
• conserved DBD 70-80 aa
• Protein structure
• Two zinc fingers constitute one separate domain
• Two ?-helices with C3-Zn-C4 N-terminally
• Disse vinkelrett på hverandre with hydrophobic
  overkrysning
• Mediates trx response to complex ekstracellular
  signals
• Evolutionary coupled to multicellular organisms
• Yeast 0 but C.elegans 233 eller 1.5 of genes
  !!
• Sequence prediksjon 90 with nuclear receptor
  DBD has potential ligand-BD
• Implies that lipophilic signal molecules have
  been important to establish communication between
  cells

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DNA-binding by nuclear receptors
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Nuclear receptors - DNA interaction

• 3D Prot-DNA structure
• glucocorticoid receptor estrogen reseptor
• Dimer in complex (monomer in solution)
• DNA interaction
• First finger binds DNA
• Second finger involved in dimerisation
• Binds to neighboring major grooves on same side
  of DNA
• Extensive phosphate contact and recognition helix
  docked into the groove
• specificity determined by 3 aa (E2, G3, A6) in
  recognition helix
• Structured dimer interphase formed upon
  DNA-binding

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GATA factors
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GATA-factors 1x Zn-C4

• Small family
• Prototype erythroid TF GATA-1 (2 fingers)
• From fungi to humans
• Structure 1.finger in nuclear receptors
• Hydrophobic DNA interphase
• Evolusjonary implicasjoner
• Early duplication of primitive finger ? divergent
  functions developed in NR

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Gal4p factors
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GAL4-related factors 1 x Zn2-C6

• GAL4-DBD
• 28aa cys-rich domain binds 2 Zn
• 26aa C-terminalt domain involv. in dimerization
• Cys-rich domain
• consensus CX2CX6CX6CX2CX6C
• A Zn-Cys cluster with shared Cys (1. and 4.)
• Two short ?-helicer with C-Zn-C N-terminalt

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GAL4-related factors 1 x Zn2-C6

• Dimerisation domain
• Monomer in solution, dimer in DNA-compleks
• In solution on ly Cys-rich motif structured
• In compleks forms two extended helix-strand
  motives
• Amfipathic helicer form a dimer-interphase in the
  complex
• DNA interaction
• contacts CGG-triplets in major groove
• C-terminal of 1. ?-helix contacts bases
• Phosphate contact via helix-strand motif
• Coiled-coil dimer-interphase at right angle to
  DNA (bZIP)
• Linker determines spacing of CGG-tripletter 11bp
  in GAL4, 6bp in PPR1

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..beyond DNA-binding
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gtDNA-binding A broader function for TFIIIA-type
???-folds

• Zif Zinc sensors
• Zif Protein-protein interaction domain
• Ikaros-homodimers
• The zinc finger protein Ikaros, which plays a
  crucial role in lymphoid differentiation, forms
  homodimers through the association of the two
  C-terminal C2H2 zinc finger motifs.
• Aiolos both homodimerizes and forms heterodimers
  with Ikaros through a two-zinc finger domain.
• GATA-1 interacts with FOG (friend of GATA)
  through a C2H2 zinc finger in GATA-1 and
  CCHC-fingers in FOG
• GATA-1 2 CCCC zinc fingers,N-terminal finger
  both DNA- and FOG-binding
• FOG 8-9 zinc fingers, mix of CCHH and CCHC
• Zif TAD (transactivation domain)
• N-terminal part ???-folded C2H2 Zif
• C-terminal part unstructured
• Zif in RNAPII (6 Zn-binding proteins)

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Recent Zif structures
Rpb10 in RNAPII
L36 ribosomal
TAZ2 In CBP
Coactivator proteins CBP and p300 contain two
copies of a zinc finger motif, termed the TAZ
finger, that are implicated in functional
interactions with numerous transcription factors
and viral oncoproteins.The TAZ2 zinc finger folds
into an unusual bundle of four helices that is
stabilized by three zinc ions, each of which is
bound to one histidine and three cysteine ligands
in an HCCC motif.
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GATA-1 like FYVE
FYVE
The FYVE domain targets cytoplasmic proteins to
specific membranes by recognition of
phosphatidylinositol 3-phosphate PI(3)P
through a highly conserved sequence motif. The
FYVE domain binds two zinc atoms, with CCCH and
CCCC coordination.
GATA-1
The structures of the FYVE and Arf-GAP domains
all contain a zinc-binding motif similar in
structure to that from GATA-1 (homology
green). (a) GATA-type zinc finger from GATA-1,
(b) FYVE domain of Vps27p